Poly(ethylene glycol) (PEG) derivatives activated with electrophilic groups are useful for coupling to nucleophilic groups, such as amino groups, of biologically active molecules. In particular, active esters and other carboxylic acid derivatives of PEG have been used to attach PEG to proteins bearing amino groups.
PEG molecules having terminal carboxymethyl groups have been described, for example, by Martinez et al., U.S. Pat. No. 5,681,567, Veronese et al., Journal of Controlled Release 10:145-154 (1989), and Buckmann et al., Makromol. Chem. 182(5): 1379-1384 (1981). U.S. Pat. No. 5,672,662 (Harris et al.) discloses PEG derivatives having a terminal propionic or butanoic acid moiety. Such carboxyl-terminated PEGs are used to prepare active esters suitable for conjugation to proteins or other molecules bearing amino groups.
However, a persistent problem associated with preparation of carboxyl-functionalized polymers has been the difficulty in obtaining the desired polymer product at a sufficiently high purity level. For example, Veronese et al. and Buckmann et al., cited above, employ a method of synthesizing mPEG carboxylic acids which comprises converting mPEG-OH to an ethyl ester of mPEG carboxylic acid, by base-catalyzed reaction of mPEG-OH with an α-halo ethyl ester, followed by base-promoted hydrolysis of the ester. However, this approach provides mPEG acids of only about 85% purity, with the main contaminant being mPEG-OH, which cannot be separated from the mPEG carboxylic acid using typical purification methods such as precipitation, crystallization or extraction. Removal of mPEG-OH requires the use of preparative ion exchange column chromatography, which is time consuming and expensive. PEG carboxylic acids obtained commercially frequently contain residual amounts of PEG-OH, which complicates the preparation of derivatives or bioconjugates based on these materials.
U.S. Pat. Nos. 5,278,303, 5,605,976 and 5,681,567 report the preparation of PEG carboxylic acids containing little or no starting material (PEG alcohol) by employing a tertiary alkyl haloacetate to prepare a tertiary alkyl ester-functionalized PEG, which is then hydrolyzed with acid, preferably trifluoroacetic acid (TFA).
Various treatises on the use of protecting groups note that tertiary alkyl esters, such as t-butyl esters, are stable to mild base hydrolysis typically used to hydrolyze primary alkyl esters, such as ethyl esters. Strong base hydrolysis could cause cleavage of carboxylic acid groups. See, for example, T. W. Greene, Protective Groups in Organic Synthesis, 3rd edition, 1999, p. 406; or P. J. Kocienski, Protecting Groups, 1994, p. 125. Accordingly, these tertiary alkyl esters are conventionally cleaved with acid, typically with TFA.
However, use of trifluoroacetic acid can result in purification and product stability problems. Trifluoroacetic acid is difficult to completely remove from the final carboxyl-functionalized polymer, particularly the amount of TFA suggested in the above-referenced patents. The presence of residual trifluoroacetic acid results in poor product stability, due to degradation of the polymer caused by acid-promoted autoxidation. See, for example, M. Donbrow, “Stability of the Polyoxyethylene Chain”, in Nonionic Surfactants: Physical Chemistry, M. J. Schick, ed., Marcel Dekker, 1987, pp. 1011 ff. This article reports that acids catalyze the formation of hydroperoxides and hydroperoxide rupture, leading to cleavage of polyoxyethylene chains.
Although U.S. Pat. No. 5,605,976 suggests distillation as a means for separating organic materials from the polymer product, even compounds with very low boiling points are difficult to remove from high molecular weight polymers using a distillation process, and the difficulty increases as the molecular weight of the polymer increases.
There is a need in the art for alternative methods for preparing carboxylic acid functionalized polymers in high yield and free from significant amounts of polymer contaminants, particularly the polymer starting material. There is also a need in the art for alternative synthesis methods that do not utilize reagents that are either difficult to remove from the final polymer product or cause product stability problems.